ABSTRACT A distinct feature of Parkinson?s Disease (PD) is the aggregation of neurotoxic ?-Synuclein (?Syn) into amyloid assemblies. Despite over two decades of intense efforts to identify inhibitors that directly interrupt the ?Syn aggregation process, no successful compounds have reached the clinic. Research on the molecular basis of PD has recently undergone a dramatic shift to focus on toxic oligomeric ?Syn and its relation to neurodegeneration. Understanding this promising new therapeutic target, a departure from research on insoluble fibrils, now requires biophysical insight about the misfolding of ?Syn monomers into the toxic oligomeric forms of the protein. Several recent studies have pointed to oxidative stress conditions as leading to the formation of highly toxic ?Syn oligomers. Thus, our first goal is to understand the molecular basis for misfolding of oxidized ?Syn. We will build on several high-impact discoveries made in the past two years that highlighted the importance of C-terminal tyrosine residues in the misfolding process. We will test a straightforward hypothesis that is based on our own recent discovery (published in Nature Chemical Biology in 2016), namely that oxidation of methionine leads to the formation of a strong non-covalent interaction with aromatic residues, including tyrosine. Our approach will provide quantitative details of the chemistry and biophysics of ?Syn, including state- of-the-art NMR measurements and computational modeling. Second, we will discover a novel set of small molecules that, for the first time, target the formation and stability of toxic, oxidized ?Syn oligomers. We will take advantage of a powerful new platform for high-throughput screening that is based on exquisitely sensitive fluorescence lifetime measurements, and test the functional efficacy of Hit compounds in neurons. These small molecules will ultimately provide a platform for our groups, in future R01-scale proposals, to launch a full-scale medicinal chemistry drug discovery campaign; but in this proposal, the Hit compounds will serve as probes to further determine (by NMR) which specific side-chain interactions with oxidized methionine drive misfolding of ?Syn. !